An extracellular matrix is composed of complicated components including collagen such as type IV collagen, and adhering glycoproteins such as proteoglycan, elastin, fibronectin, laminin, and heparan sulfate. A group of enzymes collectively named matrix metalloproteinase (hereinafter, abbreviated as MMP) having different substrate specificities are implicated in degradation of this extracellular matrix. Hitherto, as MMP, interstitial collagenase (MMP1), 72 kDa gelatinase (also referred to as type IV collagenase or gelatinase A: MMP2), stromelysin-1 (MMP3), matrilysin (MMP7), neutrophil collagenase (MMP8), 92 kDa gelatinase (also referred to as type IV collagenase or gelatinase B: MMP9), stromelysin-2 (MMP10), stromelysin-3 (MMP11), macrophage metalloelastase (MMP12), collagenase 3 (MMP13), membrane-type MMP (MT-MMP) and the like have been reported.
MMP13 which is one of the MMPs is called collagenase 3 as another name, and is much expressed in chondrocyte which is situated deep in an articular cartilage, cancer or the like. MMP13 is an extracellular matrix degrading enzyme specific for type I to type III collagen and gelatin. MMP13 has very high specificity for type II collagen which is a main extracellular matrix of cartilage, and plays an important role in a metabolism of a cartilage. Osteoarthritis is a disease of a joint accompanied with destruction of an extracellular matrix containing mainly type II collagen, and denaturation of a cartilage, and MMP13 is implicated in development of osteoarthritis through destruction of an extracellular group (see e.g. Non-patent Document 1). It is expected that, by inhibiting activity of MMP13, degradation of type II collagen which is a main extracellular matrix of cartilage is suppressed, and development of osteoarthritis is suppressed. Therefore, a neutralizing antibody of MMP13 is useful as a drug for searching, diagnosing or treating an inhibitor of a disease in which MMP13 is implicated.
MMP13 is composed of a propeptide domain, a catalytic domain, and a hemopexin clotting enzyme-like domain at a C-terminus, following a signal peptide at an N-terminus. In the catalytic domain, a zinc ion binding region to which a zinc ion essential for activity binds is present. An amino acid sequence of the catalytic domain is conserved throughout species, and the catalytic domain of human has 94%, 94%, 98%, and 96% homology to that of rat, mouse, dog and rabbit, respectively. In addition, the catalytic domain of human MMP13 has high homology to that of human MMP2, MMP1, MMP8, and MMP9, and has 77%, 73%, 73% and 73% homology thereto, respectively. There is a possibility that an antibody recognizing the catalytic domain of MMP13 has neutralizing activity, but the catalytic domain of MMP13 has high homology to the catalytic domain of human MMP2, MMP1, MMP8, and MMP9, and it is difficult to obtain a neutralizing antibody specifically recognizing MMP13.
As an antibody recognizing MMP13, there has hitherto been reported that a polyclonal antibody to recombinant MMP13 produced in Escherichia coli was made in a rabbit, in which the polyclonal antibody is used for staining a tissue of a breast cancer (see Non-patent Document 2). On the other hand, as a monoclonal antibody recognizing MMP13, a monoclonal antibody specifically reacting with both of active-type MMP13 and latent-type MMP13, a monoclonal antibody specifically reacting only with latent-type MMP13, or an antibody specifically reacting only with active-type MMP13 has been reported (see Patent Document 1). Those monoclonal antibodies do not cross-react with other matrix metal proteases, and can be used in a method of separating and quantitating latent-type MMP13 and active-type MMP13. However, there is not reported that these antibodies inhibit enzyme activity of MMP13.
Patent Document 2 describes an antibody which binds to MMP2 and MMP9, and inhibits enzyme activities thereof, and a monoclonal antibody is made using a hapten consisting of a metal ion and porphyrin, which is a structure common to a zinc ion binding region of MMP family. Therefore, this antibody has a problem in properties.
In the catalytic domain of MMP13, since the catalytic domain and the amino acid sequence of MMP2, MMP1, MMP8, MMP9 and the like are conserved as described above, it is difficult to make a neutralizing antibody which recognizes a region involved in activity of MMP13 and has MMP13-specific reactivity. Actually, there is not reported that a neutralizing monoclonal antibody inhibiting enzyme activity of MMP13 was obtained using MMP13 or a peptide thereof as an antigen.    Patent Document 1: International Publication WO 98/29560 pamphlet    Patent Document 2: International Publication WO 20000/087042 pamphlet    Non-patent Document 1: J. Clin. Invest., 1997, vol. 99, No. 7, pp. 1584-1545    Non-patent Document 2: J. Biol. Chem., 1994, vol. 269, pp. 16766-16773